CN109355406B - A kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid - Google Patents
A kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid Download PDFInfo
- Publication number
- CN109355406B CN109355406B CN201811112221.2A CN201811112221A CN109355406B CN 109355406 B CN109355406 B CN 109355406B CN 201811112221 A CN201811112221 A CN 201811112221A CN 109355406 B CN109355406 B CN 109355406B
- Authority
- CN
- China
- Prior art keywords
- detection
- seq
- primer
- kit
- label
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The kit for the detection mycobacterium tuberculosis that the invention discloses a kind of based on blood free nucleic acid, kit includes PCR reaction solution, primer, enzyme and internal standard, primer includes detection MTB target nucleotide primer and detection interior label primer, detects the nucleotide sequence of MTB target nucleotide primer as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.The detection method of kit of the present invention includes two kinds: one is based on PCR-fluorescence probe method, another kind is based on PCR-film layer analysis hybrid method；Kit of the present invention is able to achieve the detection of the hypersensitivity based on blood free nucleic acid, sputum smear is significantly improved to be negative the positive rate of tubercular, lung outer tubercular and the concurrent tuberculosis infection patient of HIV, improve the accuracy to tuberculosis infection patient's screening, detection blood sample can effectively control route of transmission, the security risk for reducing medical worker and reviewer has breakthrough clinical value to direction of medication usage lungy and control and prevention of disease.
Description
Technical field
The invention belongs to technical field of biomedical detection more particularly to a kind of detection tuberculosis based on blood free nucleic acid
The kit of mycobacteria.
Background technique
Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) abbreviation tubercle bacillus or tulase, are to draw
Send out the main pathogenic bacteria of tuberculosis (Tuberculosis, TB).Tulase is elongated slightly curved bacillus, is no spore shape
At, motionless simple unicellular bacteria.Tuberculosis is a kind of chronic infectious disease, can be propagated by air.Tuberculosis is a kind of
Seriously endanger the important diseases of human life and health: there are about 35% populations to infect tuberculosis in the whole world, and newly-increased case is super every year
8,000,000 are crossed, dies of such disease more than 2,000,000 people.China is also that tuberculosis height bears one of country, existing pulmonary tuberculosis in the world
Patient about 5,000,000, and there are about tens of thousands of people to die of the disease every year, and not only Epidemic Scope is wide by TB, also have high infection rate, high incidence and
The characteristics of high mortality.Although constantly thering is new antituberculotic to be found with the progress of medical technology, so that effectively
Control the prevalence of the disease, but increasing due to AIDS and tuberculosis double infection and multiple-drug resistance tuberculosis bacterium, this leads to this
Incremental trend is presented in the disease incidence of disease again.Therefore, timely early diagnosis is treated with accurate to prevention and control lungy and healing
It is still very crucial.
Currently, clinical diagnosis lungy is mainly according to the side such as medical history, CT, sputum smear method, Sputum culturing method and immunodiagnosis
Method, these diagnostic methods have the disadvantage that 1) traditional " goldstandard " (Sputum culturing) can identify different types of branch bar
Bacterium infection, but its period is long, sensitivity is low, is usually delayed the golden hour of patient；2) CT examination has been typically only capable to discovery
There is the patient of pulmonary lesions, can not be early diagnosed, and cannot be distinguished with other pulmonary diseases, the knot that cannot be made a definite diagnosis
Fruit；3) sputum smear is common pulmonary tuberculosis screening technology, simple and quick, but its positive rate is extremely low, the tuberculosis patient greater than 60%
It is diagnosed as smear negative；Although the tuberculosis bacterial content of smear negative patient is lower, it is still the important infection of tuberculosis
One of source easily causes and fails to pinpoint a disease in diagnosis；4) immunodiagnosis is the detection method based on specific antigen-antibody, easy to operate, but specificity
It is inadequate with sensitivity.In short, lacking a kind of highly sensitive, methods for clinical diagnosis of high specific and subsequent effective treatment
Scheme, be tuberculosis cannot effectively prevention and control and healing major reason.
Molecular diagnostic techniques have been developed in recent years diagnostic method, according to pathogen conserved sequence design specificity
Primer, probe, the reaction system for arranging in pairs or groups special and instrument carry out accurately qualitative or quantitative detection to pathogenic bacteria, have height
The characteristics of sensitivity and high specific.For the molecular diagnosis product of mycobacterium tuberculosis, now mainly there is fluorescent PCR production on the market
Product and constant-temperature amplification product, detection sensitivity usually can obtain testing result in 3~4 hours between 1~10 bacterium/ml,
It is easy quickly, but its there is also some shortcomings: 1) most of is that qualitative or Drug Resistance Detection is carried out for fresh sputum sample, and phlegm
Diagnosis of the liquid sample outside children, lung in tubercular and HIV patient is low；2) sampling is difficult, inter-sample difference is big, gained inspection
The accuracy for surveying result is low.
Blood free nucleic acid refers to small fragment DNA, RNA or the microRNA being free on outside eucaryotic cell structure in peripheral circulation blood
Equal nucleic acid molecules typically refer to the endogenous nucleic acid molecule for deriving from the release of machine apoptosis in vivo or Partial digestion；It contains
There is important hereditary information.There is only a small amount of free nucleic acid in healthy human blood, it is not easy to be detected；But in pregnant woman and tumour
The amount of free nucleic acid can be multiplied with the increase or tumor patient aggravation in pregnancy period in the blood of patient, and utilization is advanced
Nucleic acid extraction and molecular detection technology internal body point hereditary information can be interpreted.Now there are many be based on blood on the market
The molecular diagnostic techniques of liquid free nucleic acid, as using fetus dissociative DNA in maternal blood (cell free fetal DNA,
CffDNA), noninvasive Prenatal Screening (NIPT) of the tumor patient blood middle reaches from DNA (cell free tumor DNA, ctDNA)
With the biopsy of tumour liquid (liquid biopsy) technology.
According to recent document report, Nanying Che et al. and E.S.Click et al. are successively in the thoracic cavity of tuberculosis patient
Exogenous free Mycobacterium tuberculosis DNA is had found in hydrops and blood plasma, this imply that in human plasma TB cfDNA inspection
Survey will be the diagnosis target for having huge potential value, especially improve clinically sputum collecting difficulty or sputum sample detection not
The pathogen detection situation for stablizing patient, will more accurately react the severity of disease.Therefore kit of the present invention and the country are more
The design concept of Mycobacterium tuberculosis detection kit of the kind based on sputum sample is different, it is intended to research and develop a novel based on blood
The tubercle bacillus detection kit and detection method of liquid free nucleic acid.
Summary of the invention
Present invention aims to overcome that the prior art is insufficient, and provide a kind of detection tuberculosis based on blood free nucleic acid point
The kit of branch bacillus, the detection kit include two kinds: one is based on PCR-fluorescence probe method, another kind is based on PCR-
Film layer analyse hybrid method, designed and filtered out according to the conserved sequence of tubercle bacillus high sensitivity, the primer of high specific, probe,
Reaction system and response procedures realize the hypersensitivity detection based on blood free nucleic acid.
To achieve the above object, the technical scheme adopted by the invention is as follows: a kind of detection tuberculosis based on blood free nucleic acid
The kit of mycobacteria comprising PCR reaction solution, primer, enzyme and internal standard, the primer include that detection MTB target nucleotide draws
Object and detection interior label primer, nucleotide sequence such as the SEQ ID NO.1, SEQ ID of the detection MTB target nucleotide primer
NO.2, SEQ ID NO.3, shown in SEQ ID NO.4.
As an improvement of the above technical solution, the internal standard is nucleotide sequence plasmid as shown in SEQ ID NO.7, institute
Stating plasmid concentration is 2 × 104Copies/ml, nucleotide sequence such as the SEQ ID NO.5, SEQ ID of the detection interior label primer
Shown in NO.6.
As an improvement of the above technical solution, the PCR reaction solution includes following components: PCR buffer, dUTP plus
DNTP Mixture and metal cation, the enzyme are made of hot start Taq polymerase and UNG enzyme.
Preferably, in PCR reaction, the concentration of each component in PCR reaction solution are as follows: 1 × PCR buffer, 0.2~2mM
DUTP plus dNTP Mixture and 1~3mM metal cation；It is inside designated as 0.5~2 μ l, every kind of primer is 0.1~0.5 μM,
Hot start Taq polymerase is 0.5~1.5U, and (selective hydrolysis is broken the uracil sugar in the double-strand containing dU or single stranded DNA to UNG enzyme
Glycosidic bond) it is 0.05~0.5U, probe is 0.05~0.25 μM.
It as an improvement of the above technical solution, further include quality-control product, the quality-control product is by positive quality control product and negative Quality Control
Product composition, the positive quality control product are clone's bacterium solution of the target nucleotide sequences containing MTB, clone clone's bacteria concentration in bacterium solution and are
1000cfu/ml, the MTB target nucleotide sequences are as shown in SEQ ID NO.8, SEQ ID NO.9；It is described feminine gender quality-control product be
Sterile saline.
As a further improvement of the above technical scheme, kit further includes detection MTB target nucleotide fluorescence probe and inspection
Survey internal standard fluorescence probe, nucleotide sequence such as SEQ ID NO.10 and the SEQ ID of the detection MTB target nucleotide fluorescence probe
Shown in NO.11,5 ' ends of detection MTB target nucleotide fluorescence probe are marked with fluorophor, and 3 ' end labels of detection probe are quenched
Group；
The nucleotide sequence of the detection internal standard fluorescence probe detects internal standard fluorescence probe as shown in SEQ ID NO.12
5 ' ends are marked with fluorophor, 3 ' end label quenching groups of detection internal standard fluorescence probe；
Fluorophor is a kind of in FAM, HEX, VIC, NED, CY5, and quenching group is one in MGB, TAMRA, BHQ
Kind, detection MTB target nucleotide fluorescence probe and the 5 ' ends for detecting internal standard fluorescence probe are marked with different fluorophors.
As a further improvement of the above technical scheme, kit further includes hybond membrane item and developing solution, the hybond membrane
Hybridization probe is attached on item, the developing solution includes the microballoon for being connected with the second chromogenic label；
5 ' the ends for detecting the upstream primer of interior label primer are also connected with arm (C3spacer) and the first label sequence between C3 in turn
Column, the first sequence label is as shown in SEQ ID NO.15；5 ' the ends for detecting the downstream primer of interior label primer are also connected with first and show
Color marker object, the first chromogenic label can be connected with the second chromogenic label for being combined with microballoon, develop the color after microballoon enrichment；
5 ' the ends for detecting the upstream primer of MTB target nucleotide primer are also connected with arm and the second sequence label between C3 in turn,
Second sequence label is as shown in SEQ ID NO.16；5 ' the ends for detecting the downstream primer of MTB target nucleotide primer are also connected with the
One chromogenic label；
Hybridization probe includes the first hybridization probe and the second hybridization probe；The nucleotide sequence of first hybridization probe such as SEQ
Shown in ID NO.13, first hybridization probe and the first sequence label complementary pairing；The nucleotide of second hybridization probe
Sequence is as shown in SEQ ID NO.14, second hybridization probe and the second sequence label complementary pairing.
Further improvement as above-mentioned technical proposal, first chromogenic label are biotin, and described second is aobvious
Color marker object is Streptavidin, and microballoon is the latex beads that enrichment is displayed in blue.
Further improvement as above-mentioned technical proposal, the hybond membrane item include polyvinyl chloride panel, polyvinyl chloride panel
On be fixed with that cellulose nitrate is thin and blotting paper, cellulose nitrate is thin and blotting paper has overlapping, and nitrocellulose membrane is attached between the upper and lower
Every first hybridization probe and second hybridization probe.
The beneficial effects of the present invention are: the present invention provides a kind of detection mycobacterium tuberculosis based on blood free nucleic acid
Kit, which includes two kinds: one is based on PCR-fluorescence probe method, another kind is the analysis of based on PCR-film layer
Hybrid method, the kit have the advantage that
1) sample detected is blood: homogeneity is high, sampling is simple, infectiousness is low etc., can be effectively reduced healthcare givers and
The security risk coefficient of professional inspection personnel；
2) high specificity: the present invention selects two highly conserved gene order design amplimers and probe, effectively subtracts
Lack false negative result caused by due to losing genetic fragment in specific crowd；The Blast software evaluation through ncbi database is protected again
The specificity for keeping sequence, amplimer, fluorescence probe and hybridization probe, effectively prevent non-specific amplification product generate and it is more
The generation of cross reaction between kind pathogen；
3) sensibility is high: the present invention is by the testing number of a large amount of clinical sample it is demonstrated that can significantly improve sputum smear and be in
The positive rate of negative tubercular, lung outer tubercular and the concurrent tuberculosis infection patient of HIV, and to TB in people at highest risk
The accurate screening of the infected, direction of medication usage and control and prevention of disease to TB have great, breakthrough clinical value；Meanwhile to knot
The limit of identification reference material S4 (1 bacterium/ml) of core mycobacteria National reference repeat to detect 20 times positive rate (+/
+) it is 100%；
4) accuracy is high: internal control system being added in kit provided by the invention, the setting of internal control system can be supervised effectively
Sample extraction process and PCR detection process are controlled, is prevented as caused by inhibiting in sample quality, sample extraction or PCR reaction tube
False negative；Meanwhile dUTP-UNG decontamination system being added in kit, the pollution of amplified production can be effectively avoided, vacation is prevented
Positive findings；
5) quick, easy, low cost: detection kit (PCR- film layer analyses hybrid method) provided by the invention is high without dependence
The equipment such as expensive fluorescent PCR instrument, hybridization instrument carry out hybridization reaction at room temperature, and the hybridization reaction time is 5~15min, easy to operate
Quick with interpretation of result, testing cost is low, technical staff's profession is required it is low, convenient for the popularization of routine screening project；
6) practical: the present invention provides two different detection kits and methods, set for possessing different bases
Horizontal hospital is applied, alternative more, broad covered area is realized full-automatic also in combination with full-automatic detection of nucleic acids equipment
Detection.
Detailed description of the invention
Fig. 1 shows Modify to primer in the embodiment of the present invention 2, hybond membrane item label and standard color comparison card；
Fig. 2 show two kinds of kits in the embodiment of the present invention 3 (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) with
The comparing result of sputum smear method；
Fig. 3 show two kinds of kits in the embodiment of the present invention 4 (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) with
The comparing result of sputum cultivation；
Fig. 4 show two kinds of kits in the embodiment of the present invention 5 (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) with
The comparing result of TB-IGRA release reaction method；
Fig. 5 show two kinds of kits in the embodiment of the present invention 6 (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) with
The comparing result of Xpert MTB/RIF experimental method；
Fig. 6 shows the testing result of the minimum detectability of two kinds of kits in the embodiment of the present invention 7；
Fig. 7 shows the antipollution testing result of pcr amplification product of two kinds of kits in the embodiment of the present invention 7；
Fig. 8 shows the testing result of the specificity experiments of two kinds of kits in the embodiment of the present invention 7；
Fig. 9 shows that two kinds of kits are to the testing result of potential endogenous substance interference--free experiments in the embodiment of the present invention 7；
Figure 10 shows that two kinds of kits are to the detection knot of potential external source medicament residue interference--free experiments in the embodiment of the present invention 7
Fruit；
Figure 11 shows the comparing result of two kinds of kits and fluorescent PCR kit on the market in the embodiment of the present invention 8.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific experiment and attached drawing to this
Invention is described further.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.In addition, it is to be understood that using term " first ", " the in the present invention
Two " etc. describe various information, but these information should not necessarily be limited by these terms, these terms are only used to same type of information
It is distinguished from each other out.For example, without departing from the present invention, " first " information can also be referred to as " second " information,
Similar, " second " information can also be referred to as " first " information.
Embodiment 1
Kit (the PCR- fluorescence spy for detecting mycobacterium tuberculosis based on blood free nucleic acid that the present embodiment provides a kind of
The skill of handling needles) comprising PCR reaction solution, primer, enzyme, internal standard, quality-control sample, detection MTB target nucleotide fluorescence probe, detection internal standard
Fluorescence probe, positive quality control product and negative quality-control product, primer include detection MTB target nucleotide primer and detection interior label primer, inspection
Survey nucleotide sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, the SEQ ID of MTB target nucleotide primer
(2 pairs of primers) shown in NO.4；
Internal standard is for nucleotide sequence plasmid as shown in SEQ ID NO.7, and plasmid concentration is 2 × 104Copies/ml,
The nucleotide sequence of interior label primer is detected as shown in SEQ ID NO.5, SEQ ID NO.6；
PCR reaction solution includes following components: PCR buffer, dUTP plus dNTP Mixture and metal cation, enzyme
It is made of hot start Taq polymerase and UNG enzyme；In PCR reaction, the concentration of each component in PCR reaction solution are as follows: 1 × PCR buffer,
0.2~2mM dUTP plus dNTP Mixture and 1~3mM metal cation；It is inside designated as 0.5~2 μ l, every kind of primer is
0.1~0.5 μM, hot start Taq polymerase is 0.5~1.5U, and UNG enzyme is 0.05~0.5U, and every kind of fluorescence probe is 0.05~0.25 μ
M；
Positive quality control product is clone's bacterium solution of the target nucleotide sequences containing MTB, clones clone's bacteria concentration in bacterium solution and is
1000cfu/ml, MTB target nucleotide sequences are as shown in SEQ ID NO.8, SEQ ID NO.9；Negative quality-control product is sterile physiological
Salt water；
The nucleotide sequence of MTB target nucleotide fluorescence probe is detected as shown in SEQ ID NO.10, SEQ ID NO.11, inspection
5 ' the ends for surveying MTB target nucleotide fluorescence probe are marked with fluorophor, and 3 ' end labels of detection MTB target nucleotide fluorescence probe are quenched
Go out group；
The nucleotide sequence of internal standard fluorescence probe is detected as shown in SEQ ID NO.12,5 ' ends of detection internal standard fluorescence probe
It is marked with fluorophor, 3 ' end label quenching groups of detection internal standard fluorescence probe；Fluorophor be selected from FAM, HEX, VIC,
A kind of in NED, CY5, quenching group is a kind of in MGB, TAMRA, BHQ, in detection MTB target nucleotide fluorescence probe and detection
5 ' ends of mark fluorescence probe are marked with different fluorophors；Nucleic acid sequence is as shown in table 1.
Table 1
In addition, the present embodiment also provides the detection method of mentioned reagent box (PCR- fluorescence probe method) comprising following step
It is rapid:
S1) reagent prepares
According to the quantity (N) of sample to be tested and quality-control product, every part of detection reagent is prepared by following components and dosage: PCR
Reaction solution, detection MTB target nucleotide primer, detection interior label primer, detection MTB target nucleotide fluorescence probe, detection internal standard fluorescence
Probe totally 16.2 μ l, 0.3 μ l of enzyme solution, 1 μ l of internal standard；It mixes well, it is spare after brief centrifugation；
S2) the extraction and purifying (quality-control product participation extraction process) of blood free nucleic acid
A) it by blood collection into EDTA anticoagulant tube, dispenses after evenly mixing into centrifuge tube；
B) 4 DEG C, 3,000rpm, it is centrifuged 10min, upper plasma is carefully drawn, is transferred in new centrifuge tube；
C) 4 DEG C, 12,000rpm, it is centrifuged 10min, careful Aspirate supernatant is transferred in new centrifuge tube；
D) 300 μ l Proteinase K are added in 15ml centrifuge tube, add 3ml blood plasma；
E) 2.4ml Buffer BEO (containing 1.0 μ g Carrier RNA) is added, the concussion that is vortexed mixes 30s；
F) 5.4ml Buffer BEE is added into lysate by 60 DEG C of incubation 30min, and the concussion that is vortexed mixes 15~30s, ice
Upper incubation 5min；
G) LSamp Nucleic Mini adsorption column is inserted into the LCF-Connector on LSvac 20Plus, opens and inhales
Attached column lid is inserted into 20ml tube extender；
H) lysate is added in adsorption column, opens vacuum pump, after it passes through adsorption column completely, closing vacuum pump simultaneously will
Pressure is discharged to 0mbar；
I) lid for closing adsorption column takes out it from vacuum tube, is put into clean 2ml collecting pipe；
J) 600 μ l Buffer BEZ1,12,000rpm centrifugation 1min are added into adsorption column and waste liquid are outwelled, by adsorption column
It puts back in collecting pipe；
K) 750 μ l Buffer BEZ2,12,000rpm centrifugation 1min are added into adsorption column and waste liquid are outwelled, by adsorption column
It puts back in collecting pipe；
L) 750 μ l dehydrated alcohols are added in adsorption column, 12,000rpm centrifugation 1min outwell waste liquid, adsorption column is put back to
In collecting pipe；
M) 12,000rpm is centrifuged 2min, to remove residual liquid in adsorption column；
N) adsorption column is put into new 1.5ml centrifuge tube, opens lid, 10min is stored at room temperature, thoroughly to dry absorption
Remaining rinsing liquid in material；
O) the Buffer BXH (60 DEG C of preheatings) of 35 μ l is added among adsorption column film, is stored at room temperature 2min；12,000rpm
It is centrifuged 2min, elutes nucleic acid, DNA product should be stored in -20 DEG C, to prevent DNA degradation；
3) it is loaded
To the 17.5 μ l of detection reagent that the preparation of above-mentioned steps 1 is added in PCR reaction tube (N), 2 gained of above-mentioned steps is added
Each 2.5 μ l of the nucleic acid solution of sample to be tested and quality-control product, mixes gently, spare after brief centrifugation；
4) PCR amplification
3 gained PCR reaction tube (N) of above-mentioned steps is put into the sample cell of amplification instrument (real-time fluorescence quantitative PCR instrument), is pressed
The title of corresponding sequence setting sample to be tested and quality-control product, according to the form below are arranged PCR reaction condition, select fluorescence detection channel:
FAM Air conduct measurement MTB (Reporter:FAM, Quencher:None), VIC Air conduct measurement internal standard (Reporter:VIC,
Quencher:None), reference fluorescent (Passive Reference) is set as None, and reaction volume is set as 20 μ l；PCR is anti-
Answer program as shown in table 2.
Table 2
5) interpretation of result
After reaction, instrument automatically saves is automatically analyzed as a result, carrying software using instrument, can also be manual
Initial value, end value and the threshold line for adjusting baseline are analyzed, and sample Ct value is then recorded；Detection knot is judged according to the following table 3
Fruit.
Table 3
The standard of quality control is as follows: 1) the feminine gender channel quality-control product FAM should be shown without Ct value, and value≤30 internal standard Ct；2)
Value≤30 positive quality control product FAM channel C t, and value≤30 internal standard Ct；Requirements above need to meet simultaneously in same primary experiment, no
Then, this experiment is considered as invalid, need to re-start detection.
Embodiment 2
Kit (the PCR- film layer analysis for detecting mycobacterium tuberculosis based on blood free nucleic acid that the present embodiment provides a kind of
Hybrid method) comprising PCR reaction solution, primer, enzyme, internal standard, hybond membrane item, developing solution, positive quality control product and negative quality-control product,
Hybridization probe is attached on hybond membrane item；
Internal standard is nucleotide sequence plasmid as shown in SEQ ID NO.7, and plasmid concentration is 2 × 104Copies/ml, inspection
The nucleotide sequence of interior label primer is surveyed as shown in SEQ ID NO.5, SEQ ID NO.6, detects the upstream primer of interior label primer
5 ' ends are also connected with arm (C3spacer) and the first sequence label between C3, the first sequence label such as SEQ ID NO.15 institute in turn
Show；5 ' the ends for detecting the downstream primer of interior label primer are also connected with the first chromogenic label, and the first chromogenic label can be with combination
There is the second chromogenic label of microballoon to be connected, develops the color after microballoon enrichment；First chromogenic label is biotin, the second colour developing mark
Note object is Streptavidin.
Primer includes detection MTB target nucleotide primer (i.e. MTB primer pair) and above-mentioned detection interior label primer, detects MTB target
The nucleic acid sequence of nucleotide primer is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4；Inspection
5 ' the ends for surveying the upstream primer of MTB target nucleotide primer are also connected with arm and the second sequence label between C3, the second label sequence in turn
Column are as shown in SEQ ID NO.16；5 ' the ends for detecting the downstream primer of MTB target nucleotide primer are also connected with the first revealing label
Object；
PCR reaction solution includes following components: PCR buffer, dUTP plus dNTP Mixture and metal cation, enzyme
It is made of hot start Taq polymerase and UNG enzyme；In PCR reaction, the concentration of each component in PCR reaction solution are as follows: 1 × PCR buffer,
0.2~2mM dUTP plus dNTP Mixture and 1~3mM metal cation；It is inside designated as 0.5~2 μ l, every kind of primer is
0.1~0.5 μM, hot start Taq polymerase is 0.5~1.5U, and UNG enzyme is 0.05~0.5U；
Quality-control product is made of positive quality control product and negative quality-control product, and positive quality control product is gram of the target nucleotide sequences containing MTB
Grand bacterium solution, cloning clone's bacteria concentration in bacterium solution is 1000cfu/ml, MTB target nucleotide sequences such as SEQ ID NO.8, SEQ ID
Shown in NO.9；Negative quality-control product is sterile saline；
Developing solution includes the microballoon for being connected with the second chromogenic label, developing solution contained substance it is final concentration of: 100mM
HEPPS-Hcl buffer (HEPPS, 4- hydroxyethyl piperazine propane sulfonic acid), pH 8.0；1M sodium chloride, 0.4mM EDTA solution, pH
8.0,1%latex microballoons.
Hybridization probe includes the first hybridization probe and the second hybridization probe, the sequence of the first hybridization probe such as SEQ ID
Shown in NO.13, the sequence of the second hybridization probe is as shown in SEQ ID NO.14；Hybond membrane item the preparation method comprises the following steps: by nitric acid fibre
It ties up plain film (4.6cm × 20cm) to be attached in PVC board (6cm × 20cm), then blotting paper (2cm × 20cm) is pasted on PVC board
On, there is 0.6cm overlapping with nitrocellulose filter；2 kinds of probes are drawn on nitrocellulose filter with the concentration spray of 20ng/ μ l
(Probe:200ng/cm, Hp1, Hp2), and (the first hybridization probe is second using the red pad-ink of spray stroke as position line differentiation
The top of hybridization probe)；The semi-finished product PVC board made is placed in 37 DEG C of oven dried overnights, is finally cut into (2mm × 6cm)
Finished product PAS film item, as shown in Figure 1；Meanwhile nucleic acid sequence is as shown in table 1.
In addition, the present embodiment also provides the detection method of mentioned reagent box (PCR- film layer analyse hybrid method), successively include with
Lower step:
S1) reagent prepares
According to the quantity (N) of sample to be tested and quality-control product, every part of detection reagent is prepared by following components and dosage: PCR
13 μ l of reaction solution, internal standard and primer totally 4.2 μ l, 0.3 μ l of enzyme solution；It mixes well, spare reagent prepares after brief centrifugation；
S2) the extraction and purifying (quality-control sample participation extraction process) of blood free nucleic acid
A) it by blood collection into EDTA anticoagulant tube, dispenses after evenly mixing into centrifuge tube；
B) 4 DEG C, 3,000rpm, it is centrifuged 10min, upper plasma is carefully drawn, is transferred in new centrifuge tube；
C) 4 DEG C, 12,000rpm, it is centrifuged 10min, careful Aspirate supernatant is transferred in new centrifuge tube；
D) 300 μ l Proteinase K are added in 15ml centrifuge tube, add 3ml blood plasma；
E) 2.4ml Buffer BEO (containing 1.0 μ g Carrier RNA) is added, the concussion that is vortexed mixes 30s；
F) 5.4ml Buffer BEE is added into lysate by 60 DEG C of incubation 30min, and the concussion that is vortexed mixes 15~30s, ice
Upper incubation 5min；
G) LSamp Nucleic Mini adsorption column is inserted into the LCF-Connector on LSvac 20Plus, opens and inhales
Attached column lid is inserted into 20ml tube extender；
H) lysate is added in adsorption column, opens vacuum pump, after it passes through adsorption column completely, closing vacuum pump simultaneously will
Pressure is discharged to 0mbar；
I) lid for closing adsorption column takes out it from vacuum tube, is put into clean 2ml collecting pipe；
J) 600 μ l Buffer BEZ1,12,000rpm centrifugation 1min are added into adsorption column and waste liquid are outwelled, by adsorption column
It puts back in collecting pipe；
K) 750 μ l Buffer BEZ2,12,000rpm centrifugation 1min are added into adsorption column and waste liquid are outwelled, by adsorption column
It puts back in collecting pipe；
L) 750 μ l dehydrated alcohols are added in adsorption column, 12,000rpm centrifugation 1min outwell waste liquid, adsorption column is put back to
In collecting pipe；
M) 12,000rpm is centrifuged 2min, to remove residual liquid in adsorption column；
N) adsorption column is put into new 1.5ml centrifuge tube, opens lid, 10min is stored at room temperature, thoroughly to dry absorption
Remaining rinsing liquid in material；
O) the Buffer BXH (60 DEG C of preheatings) of 35 μ l is added among adsorption column film, is stored at room temperature 2min；12,000rpm
It is centrifuged 2min, elutes nucleic acid, DNA product should be stored in -20 DEG C, to prevent DNA degradation；
3) it is loaded
To the 17.5 μ l of detection reagent that the preparation of above-mentioned steps 1 is added in PCR reaction tube (N), 2 gained of above-mentioned steps is added
Each 2.5 μ l of the nucleic acid solution of sample to be tested and quality-control product, mixes gently, spare after brief centrifugation；
4) PCR amplification
3 gained PCR reaction tube (N) of above-mentioned steps is put into the sample cell of amplification instrument (common gene-amplificative instrament), is reacted
It is as shown in table 4 that volume is set as 20 μ l, PCR response procedures.
Table 4
5) chromatography hybridization
After reaction, by PCR reaction tube (N) brief centrifugation, it is molten that 20 μ l colour developing is added in equal volume in Biohazard Safety Equipment
Liquid after mixing well, is inserted into PAS film item in PCR reaction tube (N), can interpretation result after the reaction of 5~15min quick hybridization.
6) interpretation of result
The band relative position that can be found in region shown in Fig. 1 Plays colorimetric card carries out qualitative interpretation, also can refer to following table
5, preservation of taking pictures is carried out to colour developing result.
Table 5
| As a result | Quality control region | Detection zone | Interpretation |
| 1 | With/without blue bands | There are blue bands | It is positive |
| 2 | There are blue bands | Without blue bands | It is negative |
The standard of quality control is as follows: 1) 1 blue bands should occur in negative quality-control product, be located at internal standard (IC line), and examine
Surveying area (T line) does not have band or intensity < L4；2) 2 blue bands should occur in positive quality control product, be located at internal standard (IC line)
With detection zone (T line), and intensity >=L4；If 3) pattern detection is feminine gender, internal standard (IC line) band intensity answers >=L2, and sample has
Effect；If 4), pattern detection is the positive, internal standard (IC line) band intensity can by force can be weak, or even without band, sample is effective；It wants above
Asking in same primary experiment while need to meet, and otherwise, it is invalid that this experiment is considered as, and need to re-start detection.
Embodiment 3
The present embodiment detects clinical sample using Examples 1 to 2 kit and sputum smear method, and clinical sample is practical
Situation are as follows: tuberculosis cases 103, extrapulmonary tuberculosis example 26, negative case 43, total cases are 172；The present embodiment
Two kinds of kits (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) measurement results as shown in Fig. 2, and with sputum smear method
It compares, comparing result is as shown in table 6；Note: it is sputum smear testing result that table 6 is endways, which to be seen, sees it is two kinds of examinations of the present invention sidewards
Agent box testing result；Below table view is similar.
Table 6
As shown in table 6, the overall positive rate and negative rate checked using sputum smear method is respectively 20.93% (36/172)
With 79.07% (136/172), lunger, the outer tubercular of lung positive rate be respectively 20.93% (36/172), 0%
(0/172)；Wherein, 12 pulmonary tuberculosis positive cases, follow up according to follow-up clinical, are diagnosed as NTM (non-tuberculous mycobacteria) sense
Dye；Use clinical diagnosis result for goldstandard, through counting, the false positive rate of sputum smear method is 27.91% (12/43), false negative
Rate is 81.4% (105/129), and diagnostic accordance rate is 31.98% (55/172), though this method is easy to operate, is suitable for common screening,
But it cannot distinguish between NTM cases of infection, it is difficult to detect to tuberculosis outside lung, and sensibility and specificity is too poor, and diagnostic accordance rate is low.
However, two kinds of kit test results of the invention are consistent: the overall positive rate and feminine gender of 172 blood samples
Rate is respectively 75% (129/172) and 25% (43/172), and the positive rate of tubercular is respectively outside lunger, lung
59.88% (103/172), 15.12% (26/172)；Use clinical diagnosis result for goldstandard, through counting, the vacation of the kit
Positive rate is 0% (0/43), false negative rate is 0% (0/129), diagnostic accordance rate is 100% (172/172), effective to distinguish
MTB infection and NTM cases of infection significantly improve the diagnosis of sputum smear negative tuberculosis case and the outer tubercular of lung, improve TB
The accuracy rate and coincidence rate of diagnosis.
Embodiment 4
The present embodiment detects clinical sample using Examples 1 to 2 kit and Sputum culturing method, and clinical sample is practical
Situation are as follows: tuberculosis cases 126, extrapulmonary tuberculosis example 39, negative case is 48, and total cases are 213；This implementation
Two kinds of kits of example (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) measurement result as shown in figure 3, and with Sputum culturing method
Method compares, and comparing result is as shown in table 7.
Table 7
As shown in table 7, Sputum culturing method detection overall positive rate and negative rate be respectively 29.58% (63/213) and
70.42% (150/213), lunger, the outer tubercular of lung positive rate be respectively 28.64% (61/213), 0.94%
(2/213)；Use clinical diagnosis result for goldstandard, through counting, this method false positive rate is 0% (0/48), and false negative rate is
61.82% (102/165), diagnostic accordance rate are 52.11% (111/213).Though this method specificity is preferably, detection cycle mistake
Long, it is difficult to diagnose to tubercular outside lung, and sensibility is poor, easily causes and fails to pinpoint a disease in diagnosis.
However, two kinds of kit test results of the invention are consistent: the overall positive rate and feminine gender of 213 blood samples
Rate is respectively 77.46% (165/213) and 22.54% (48/213), the positive rate difference of tubercular outside lunger, lung
For 59.15% (126/213), 18.31% (39/213)；Use clinical diagnosis result for goldstandard, through counting, the false sun of this method
Property rate be 0% (0/48), false negative rate be 0% (0/165), diagnostic accordance rate be 100% (213/213), kit of the present invention
Detection cycle be obviously shortened, operate easier, reduce the false negative result as caused by tulase is dead or vigor is low, show
The positive rate for improving Sputum culturing feminine gender tuberculosis case and the outer tubercular of lung is write, the accuracy rate and coincidence rate of TB diagnosis are improved.
Embodiment 5
The present embodiment detects clinical sample using Examples 1 to 2 kit and Sputum culturing method, and clinical sample is practical
Situation are as follows: tuberculosis cases 24, HIV+TB case 37, extrapulmonary tuberculosis example 15, negative case is 13, total cases
It is 89；Two kinds of kits of the present embodiment (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) measurement result as shown in figure 4,
And compared with TB-IGRA release reaction method, comparing result is as shown in table 8.
Table 8
As shown in table 8, TB-IGRA discharges the overall positive rate of reaction method detection and negative rate is respectively 56.18% (50/
89) and 43.82% (39/89), lunger, HIV+TB patient, the outer tubercular of lung positive rate be respectively 25.84%
(23/89), 15.73% (14/89), 14.61% (13/89)；Use clinical diagnosis result for goldstandard, through counting, this method
False positive rate is 46.15% (6/13), and false negative rate is 42.11% (32/76), and diagnostic accordance rate is 57.3% (51/89), this
Method is high for the positive rate of Lung infection tulase (87.5%, 21/24), it was reported that gamma interferon it is quantitative it is higher with it is movable
Property tuberculosis have certain correlation, can not identify completely latent tuberculosis bacterium infection and active tuberculosis, can distinguish MTB sense
Dye is infected with NTM, can also be had to tuberculosis infection outside lung higher positive rate (80%, 12/15), but since some cases are deposited
In immune deficiency or received immunization therapy, the intracorporal immune response inferior capabilities of machine, for the concurrent active tuberculosis infection of HIV
The positive rate of patient is obvious relatively low (29.73%, 11/37), easily causes mistaken diagnosis.
However, two kinds of kit test results of the invention are consistent: the overall positive rate and feminine gender of 89 blood samples
Rate is respectively 85.39% (76/89) and 14.61% (13/89), lunger, HIV+TB patient, the outer tubercular of lung sun
Property rate is respectively 26.97% (24/89), 41.57% (37/89), 16.85% (15/89)；Use clinical diagnosis result for gold mark
Standard, through counting, this method false positive rate is 0% (0/13), and false negative rate is 0% (0/76), and diagnostic accordance rate is 100% (89/
89).The present invention uses free nucleic acid in blood to be avoided that the defect of immunodiagnosis as detection target, significantly improve HIV simultaneously
The positive rate for sending out tuberculosis infection case combines hypersensitivity and specificity to tuberculosis outside pulmonary tuberculosis and lung, improves
The accuracy rate and coincidence rate of TB diagnosis.
Embodiment 6
The present embodiment detects clinical sample using Examples 1 to 2 kit and Xpert MTB/RIF experimental method,
Clinical sample actual conditions are as follows: tuberculosis cases 31, HIV+TB case 17, extrapulmonary tuberculosis example 18, negative case are
55, total cases are 121；Two kinds of kits of the present embodiment (PCR- fluorescence probe method and PCR- film layer analyse hybrid method) measurement
As a result as shown in figure 5, and compared with Xpert MTB/RIF experimental method, comparing result is as shown in table 9.
Table 9
As shown in table 9, it is respectively using the overall positive rate and negative rate of Xpert MTB/RIF experimental method detection
38.02% (46/121) and 61.98% (75/121), lunger, HIV+TB patient, the outer tubercular of lung positive rate point
It Wei not 27.27% (33/121), 6.61% (8/121), 4.13% (5/121)；Use clinical diagnosis result for goldstandard, through uniting
Meter, this method false positive rate are 7.27% (4/55), and false negative rate is 36.36% (24/66), diagnostic accordance rate 76.86%
(93/121), this method is easy quickly, automatically detects, and can detect to tulase drug resistance situation, with higher sensitive
Degree and specificity are the molecular diagnosis methods more generally used at present, but for certain special cases, the acquisition of sputum is relatively tired
Difficulty, poor to the detection sensitivity of expectoration phlegm or induction of sputum, gastric juice equal samples such as tubercular outside children TB patient or lung, inspection
It is poor to survey precision, therefore has some disadvantages.
However, two kinds of kit test results of the invention are consistent: the overall positive rate and feminine gender of 121 blood samples
Rate is respectively 54.55% (66/121) and 45.45% (55/121), lunger, HIV+TB patient, lung outer tubercular
Positive rate is respectively 25.62% (31/121), 14.05% (17/121), 14.88% (18/121)；Using clinical diagnosis result
For goldstandard, through counting, this method false positive rate is 0% (0/55), and false negative rate is 0% (0/66), and diagnostic accordance rate is
100% (121/121).Since sample to be tested is blood, the case where capable of effectively avoiding above-mentioned special case sampling difficulty, sample
Homogeneity is more preferable, and utilizes advanced free nucleic acid extractive technique, good through testing result precision, for above-mentioned special case
There is better sensibility and specificity, improves the accuracy rate and coincidence rate of TB diagnosis.
In short, showing two kinds of kits (PCR- fluorescence probe method and PCR- films of the invention according to above-mentioned 4 groups of clinical datas
Chromatography hybridization method) have fabulous sensibility and specificity, compared with existing detection means, can significantly improve sputum smear,
The positive detection of the concurrent tuberculosis infection patient of positive rate and HIV of Sputum culturing feminine gender tubercular and the outer tubercular of lung
Rate solves the problems, such as that special case sputum collecting sample is difficult and homogeneity is poor, and detection cycle is short, easy to be quick, and not with
Cross reaction occurs for the pulmonary disease of non-tuberculosis, reaches 100% with the coincidence rate of clinical diagnosis result.
Embodiment 7
To prove reasonability and feasibility of the invention, following Performance Evaluation is carried out to kit and method and has been studied:
1) minimum detectability experiment (sensitivity experiment)
The tuberculosis branch bar that National Institute for Food and Drugs Control is provided according to above-described embodiment 1,2 kits and method
Bacterium PCR detection kit carries out 20 repetitions with the limit of identification reference material S4 (1 bacterium/ml) in National reference and detects,
Its positive rate (+/+) it is 100% (20/20), it is very high to illustrate that detection kit and method provided by the present invention have
Sensitivity (a bacterium/ml), hence it is evident that better than common fluorescence PCR method and hybrid method, as a result as shown in Figure 6.
2) the prevention experiment of PCR product pollution
In the subsequent processing to PCR product, be easy to produce aerosol phenomenon (pcr amplification product containing positive sample,
Concentration is generally below 1 × 103Copies/ml), cause laboratory environment to pollute, certain false positive wind is brought to subsequent experimental
Danger, therefore the measure of setting prevention PCR product pollution is critically important in PCR system.UNG- is provided in kit of the present invention
DUTP decontamination system using the U-DNA amplified production in UNG enzymatic hydrolysis reaction system, while passing through high-temperature inactivation UNG enzyme,
The production of new round PCR product is not influenced, to have the function that Pollution protection.
Following sample to be tested is detected according to above-described embodiment 1,2 kits and method, with two groups of different reactions
System (the 1st group: enzyme containing UNG, the 2nd group: being free of UNG enzyme) compares experiment, and measurement UNG enzyme prevents U-DNA amplified production
Pollution effects, the preparation of sample to be tested and testing result are as shown in table 10.
Table 10
As shown in table 10 and Fig. 7, UNG-dUTP decontamination system is added in kit of the present invention, can effectively prevent U-
The pollution of DNA cloning product.
3) specificity experiments
The tuberculosis branch bar that National Institute for Food and Drugs Control is provided according to above-described embodiment 1,2 kits and method
Negative reference product (mycobacterium avium, soil mycobacteria, Amur branch bar in bacterium PCR detection kit National reference
Bacterium, Asia mycobacteria, Mycobacterium scrofulaceum, mycobacterium gordonae, tortoise purulence mycobacteria, is accidentally divided mycobacterium kansasii
Branch bacillus, Mycobacterium graminis, Nocardia brasiliensis, Beijing corynebacterium, pneumococcus, legionella pneumophilia, pertussis Bordetella
Bacterium) and mycoplasma pneumoniae, chlamydia pneumoniae, Respiratory Syncytial Virus(RSV) and influenza virus equal samples extract and detect,
Negative recall rate (-/-) it is 100% (19/19), illustrate that detection kit and method provided by the present invention have very high spy
With other common pathogenic organisms of respiratory tracts cross reaction will not occur for the opposite sex；Testing result is as shown in Figure 8.
4) to the anti-interference of potential endogenous substance
According to above-described embodiment 1,2 kits and method to containing the potential endogenous substance of 5% dosage (15%EDTA solution,
120g/L hemoglobin, 1mM triglycerides) clinical blood sample extract and detect.As shown in figure 9, testing result is equal
It is consistent with Normal group and clinical diagnosis result, illustrate kit provided by the present invention and method to potential endogenous substance
Strong interference immunity.
5) to the anti-interference of potential external source medicament residue
According to above-described embodiment 1,2 kits and method to the potential external source drug containing 5% dosage medicine related levels
Concentration (10 isoniazid μ g/ml, 250 μ g/ml rifampins, 10 μ g/ml lavo-ofloxacins, 50 μ g/ml ethambutols, 250 μ g/ml
Pyrazinamide) clinical blood sample extract and detect.As shown in Figure 10, testing result with Normal group and clinic
Diagnostic result is consistent, illustrates kit provided by the present invention and method to the latent strong interference immunity to external source medicament residue.
Embodiment 8
The present embodiment uses the cfDNA sample (N=50) of gained clinical blood sample in above-described embodiment 3, and sample comes
From the Pulmonary Disease patients of tubercular outside the lunger, HIV+TB patient, lung clinically made a definite diagnosis and exclusion tuberculosis, sample
This number is respectively 9,11,13,17, and use preferable mycobacterium tuberculosis fluorescence PCR kit on the market is strictly pressed
It is detected according to shop instruction；Testing result is as shown in table 11 and Figure 11.
Table 11
As shown in table 11, the overall positive rate of certain producer's mycobacterium tuberculosis fluorescence PCR kit and negative rate are respectively
12% (6/50) and 88% (44/50)；Use clinical diagnosis result for goldstandard, through counting, this method diagnostic accordance rate is
46% (23/50).
However, two kinds of kit test results of the invention are consistent: overall positive rate and negative rate are respectively 66%
(33/50) and 34% (17/50)；Use clinical diagnosis result for goldstandard, through counting, this method diagnostic accordance rate is 100%
(50/50), the specificity of detection kit and method of the present invention and sensibility are significantly better than fluorescent PCR detection at present on the market
Kit.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and
Range.
Sequence table
<110>Guangzhou Bao Chuan Bioisystech Co., Ltd
<120>a kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid
<130> 2018.9.12
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial synthesized sequence
<400> 1
cgtacaaggc cttcgattgg 20
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized sequence
<400> 2
gctcagttca ccttgcacaa 20
<210> 3
<211> 19
<212> DNA
<213>artificial synthesized sequence
<400> 3
gtaggcgtcg gtgacaaag 19
<210> 4
<211> 19
<212> DNA
<213>artificial synthesized sequence
<400> 4
ggaccaccag cacctaacc 19
<210> 5
<211> 18
<212> DNA
<213>artificial synthesized sequence
<400> 5
gagtatgcct gccgtgtg 18
<210> 6
<211> 18
<212> DNA
<213>artificial synthesized sequence
<400> 6
aatccaaatg cggcatct 18
<210> 7
<211> 110
<212> DNA
<213>artificial synthesized sequence
<400> 7
gagtatgcct gccgtgtgaa ccatgtgact ttgtcacagc ccaagatagt taagtgggat 60
cgagacatgt aagcagcatc atggaggttt gaagatgccg catttggatt 110
<210> 8
<211> 119
<212> DNA
<213>artificial synthesized sequence
<400> 8
cgtacaaggc cttcgattgg gaccaggcct atcgcaagcc aatcacctat gacacgctgt 60
ggcaggctga caccgatccg ctgccagtcg tcttccccat tgtgcaaggt gaactgagc 119
<210> 9
<211> 96
<212> DNA
<213>artificial synthesized sequence
<400> 9
gtaggcgtcg gtgacaaagg ccacgtaggc gaaccctgcc caggtcgaca cataggtgag 60
gtctgctacc cacagccggt taggtgctgg tggtcc 96
<210> 10
<211> 20
<212> DNA
<213>artificial synthesized sequence
<400> 10
cctatcgcaa gccaatcacc 20
<210> 11
<211> 22
<212> DNA
<213>artificial synthesized sequence
<400> 11
cacgtaggcg aaccctgccc ag 22
<210> 12
<211> 20
<212> DNA
<213>artificial synthesized sequence
<400> 12
tgactttgtc acagcccaag 20
<210> 13
<211> 23
<212> DNA
<213>artificial synthesized sequence
<400> 13
cgagatcggt ggttacttag att 23
<210> 14
<211> 21
<212> DNA
<213>artificial synthesized sequence
<400> 14
gcaaccttag agcaaggtca a 21
<210> 15
<211> 23
<212> DNA
<213>artificial synthesized sequence
<400> 15
gctctagcca ccaatgaatc taa 23
<210> 16
<211> 21
<212> DNA
<213>artificial synthesized sequence
<400> 16
cgttggaatc tcgttccagt t 21
Claims (8)
1. a kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid, which is characterized in that reacted including PCR
Liquid, primer, enzyme and internal standard, the primer include detection MTB target nucleotide primer and detection interior label primer, the detection MTB target
The nucleotide sequence of nucleotide primer is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.
2. kit as described in claim 1, which is characterized in that the internal standard is nucleotide sequence such as SEQ ID NO.7 institute
The plasmid shown, the plasmid concentration are 2 × 104Copies/ml, the nucleotide sequence such as SEQ ID of the detection interior label primer
Shown in NO.5, SEQ ID NO.6.
3. kit as described in claim 1, which is characterized in that the PCR reaction solution includes following components: PCR buffer,
DUTP plus dNTP Mixture and metal cation, the enzyme are made of hot start Taq polymerase and UNG enzyme.
4. kit as claimed in any one of claims 1 to 3, which is characterized in that further include quality-control product, the quality-control product is by sun
Property quality-control product and negative quality-control product composition, the positive quality control product be clone's bacterium solution of the target nucleotide sequences containing MTB, clone bacterium solution
Middle clone's bacteria concentration is 1000cfu/ml, and the MTB target nucleotide sequences are as shown in SEQ ID NO.8, SEQ ID NO.9；Institute
Stating negative quality-control product is sterile saline.
5. kit as claimed in claim 4, which is characterized in that further include detection MTB target nucleotide fluorescence probe and detection
Internal standard fluorescence probe, nucleotide sequence such as SEQ ID NO.10 and the SEQ ID of the detection MTB target nucleotide fluorescence probe
Shown in NO.11,5 ' ends of detection MTB target nucleotide fluorescence probe are marked with fluorophor, and 3 ' end labels of detection probe are quenched
Group；
The nucleotide sequence of the detection internal standard fluorescence probe is as shown in SEQ ID NO.12,5 ' ends of detection internal standard fluorescence probe
It is marked with fluorophor, 3 ' end label quenching groups of detection internal standard fluorescence probe；
Fluorophor is a kind of in FAM, HEX, VIC, NED, CY5, and quenching group is a kind of in MGB, TAMRA, BHQ, inspection
5 ' the ends for surveying MTB target nucleotide fluorescence probe and detection internal standard fluorescence probe are marked with different fluorophors.
6. kit as claimed in claim 4, which is characterized in that further include hybond membrane item and developing solution, the hybond membrane item
On be attached with hybridization probe, the developing solution includes the microballoon for being connected with the second chromogenic label；
5 ' the ends for detecting the upstream primer of interior label primer are also connected with arm and the first sequence label between C3, the first sequence label in turn
As shown in SEQ ID NO.15；5 ' the ends for detecting the downstream primer of interior label primer are also connected with the first chromogenic label, and first is aobvious
Color marker object can be connected with the second chromogenic label for being combined with microballoon, develop the color after microballoon enrichment；
The 5 ' of the upstream primer of detection MTB target nucleotide primer, which are held, is also connected with arm and the second sequence label between C3 in turn, and second
Sequence label is as shown in SEQ ID NO.16；5 ' the ends for detecting the downstream primer of MTB target nucleotide primer are also connected with first and show
Color marker object；
Hybridization probe includes the first hybridization probe and the second hybridization probe；The nucleotide sequence of first hybridization probe such as SEQ ID
Shown in NO.13, first hybridization probe and the first sequence label complementary pairing；The nucleotide sequence of second hybridization probe
As shown in SEQ ID NO.14, second hybridization probe and the second sequence label complementary pairing.
7. kit as claimed in claim 6, which is characterized in that first chromogenic label be biotin, described second
Chromogenic label is Streptavidin, and microballoon is the latex beads that enrichment is displayed in blue.
8. kit as claimed in claim 6, which is characterized in that the hybond membrane item includes polyvinyl chloride panel, polyvinyl chloride
It is fixed with that cellulose nitrate is thin and blotting paper on plate, cellulose nitrate is thin and blotting paper has overlapping, and nitrocellulose membrane is attached with up and down
First hybridization probe and second hybridization probe at interval.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811112221.2A CN109355406B (en) | 2018-09-21 | 2018-09-21 | A kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811112221.2A CN109355406B (en) | 2018-09-21 | 2018-09-21 | A kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109355406A CN109355406A (en) | 2019-02-19 |
| CN109355406B true CN109355406B (en) | 2019-07-09 |
Family
ID=65351457
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811112221.2A Active CN109355406B (en) | 2018-09-21 | 2018-09-21 | A kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109355406B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988867B (en) * | 2019-03-13 | 2021-03-23 | 广州市宝创生物技术有限公司 | Kit for detecting respiratory pathogens of community-acquired pneumonia |
| CN110714090B (en) * | 2019-12-03 | 2023-08-22 | 郑州安图生物工程股份有限公司 | Kit for detecting free nucleic acid of blood flow infection pathogen in blood plasma |
| CN111073984A (en) * | 2019-12-30 | 2020-04-28 | 江苏猎阵生物科技有限公司 | Kit and method for detecting target nucleic acid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105218648A (en) * | 2015-07-10 | 2016-01-06 | 邓林红 | Negre antigen, nucleotide sequence and application thereof |
| CN105296661A (en) * | 2015-12-02 | 2016-02-03 | 北京市结核病胸部肿瘤研究所 | Kit for diagnosing tuberculosis by detecting free nucleic acid and application of kit |
| CN106967828A (en) * | 2017-05-17 | 2017-07-21 | 上海默礼生物医药科技有限公司 | A kind of nucleic acid extracting reagent, kit and its method for being used to detect low-copy mycobacterium tuberculosis |
-
2018
- 2018-09-21 CN CN201811112221.2A patent/CN109355406B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105218648A (en) * | 2015-07-10 | 2016-01-06 | 邓林红 | Negre antigen, nucleotide sequence and application thereof |
| CN105296661A (en) * | 2015-12-02 | 2016-02-03 | 北京市结核病胸部肿瘤研究所 | Kit for diagnosing tuberculosis by detecting free nucleic acid and application of kit |
| CN106967828A (en) * | 2017-05-17 | 2017-07-21 | 上海默礼生物医药科技有限公司 | A kind of nucleic acid extracting reagent, kit and its method for being used to detect low-copy mycobacterium tuberculosis |
Non-Patent Citations (2)
| Title |
|---|
| MG545261.1;NCBI;《GenBank》;20180902;第1页 |
| 结核分枝杆菌IS6110和IS1081微滴数字PCR检测体系的建立和应用;李自慧等;《北京医学》;20180430;第40卷(第4期);摘要 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109355406A (en) | 2019-02-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110904225B (en) | Combined marker for liver cancer detection and application thereof | |
| Ismoilovich | Tuberculosis Diagnostics with Modern Solutions (Literature Review) | |
| CN109777874A (en) | It is a kind of suitable for ductal adenocarcinoma of pancreas diagnosis and Index for diagnosis blood plasma excretion body miRNA marker and application | |
| CN109355406B (en) | A kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid | |
| CN107955836A (en) | For the primer pair of lung cancer related gene SHOX2 DNA methylation assays, kit and method | |
| JP2021502069A5 (en) | ||
| CN107164526A (en) | A kind of method for visualizing genechip detection Pathogen of Lung Infection | |
| AU2020102244A4 (en) | Primer group, kit and detection method for rapidly detecting carbapenemases genes | |
| CN109055555A (en) | A kind of lung cancer transfer diagnosis marker and its kit and application in early days | |
| CN108048589A (en) | The Real-time PCR specific primers and probe and detection kit and detection method of ox two-pressure humidity generator | |
| CN107475389A (en) | For detecting primer sets, kit and the method for mycoplasma pneumoniae | |
| CN104694623A (en) | Plasma miRNA marker for diagnosis of lung cancer and application | |
| CN110241256A (en) | A kind of kit of multiple quantitative detection Epstein-Barr virus and cytomegalovirus | |
| JP7187081B2 (en) | Methods for early diagnosis and post-treatment monitoring of breast cancer using liquid biopsy multiplex oncogene biomarkers | |
| CN107513494A (en) | A kind of detection of nucleic acids card and its application method | |
| CN114438238B (en) | Primer for detecting infectious endocarditis pathogen and digital PCR kit | |
| WO2017092483A1 (en) | Kit for diagnosing tuberculosis through detecting free nucleic acid and use thereof | |
| CN110331208A (en) | Application of the molecular target in adenocarcinoma of lung Combining diagnosis | |
| CN110305953A (en) | The system for detecting miRNA expression quantity distinguishes the application in tubercular meningitis and viral meningitis product in preparation | |
| CN109735612A (en) | The biomolecule marker and its kit of Kawasaki disease coronary aneurysm complication | |
| CN101760518A (en) | Method for extracting live bacteria RNA in Mycobacterium tuberculosis and detection kit thereof | |
| CN111455094B (en) | Composition, kit, application and method for detecting deep infection fungi | |
| CN111733227B (en) | Molecular marker circRNA for diagnosing idiopathic optic neuritis, kit and application | |
| CN114686586B (en) | Molecular marker composition for diagnosing tuberculous pleurisy and application thereof | |
| CN110527721A (en) | A kind of oldness tuberculosis marker and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |